Type I interferons (IFNs) are essential components of the innate immune system. This study characterized the distinct IFN sensitivities of two closely related Semliki Forest virus (SFV) strains in cell culture. The virulent L10 strain was derived from the original virus isolate by propagation in mice. In contrast, the avirulent SFV strain, designated V42, was derived from an earlier passage of the original virus isolated from mosquitoes. The virulent L10 strain produced a cytopathic effect (CPE) in IFN-treated cells and the production of infectious virus was only two orders of magnitude lower compared with untreated cells. In contrast, the avirulent V42 exerted no CPE in IFN-treated cells and production of infectious virus was four orders of magnitude lower compared with untreated cells. The reduced CPE in IFN-treated cells infected with the avirulent V42 strain was due to inhibition of productive infection and not to reduced cell death. The virulent L10 strain synthesized less genomic RNA but more non-structural proteins than the avirulent V42 strain, suggesting more efficient translation of the L10 genomic RNA. Using a cell line unable to produce IFN, it was shown that the reduced susceptibility of the L10 strain to the action of IFN was not due to reduced IFN induction. Hence, the reduced susceptibility of the virulent L10 strain to the action of IFN allows it to overcome the established IFN-induced antiviral state of the cell, thereby increasing its virulence.